QC Report


general
Report generated at2025-02-22 03:03:27
TitleLiver
DescriptionATAC-seq data from Liu et al. liver data processed in 2025
Pipeline versionv2.2.0
Pipeline typeatac
Genomemm10
Alignerbowtie2
Sequencing endedness{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak callermacs2

Alignment quality metrics


SAMstat (raw unfiltered BAM)

rep1rep2
Total Reads137766628138223394
Total Reads (QC-failed)00
Duplicate Reads00
Duplicate Reads (QC-failed)00
Mapped Reads134745158135277039
Mapped Reads (QC-failed)00
% Mapped Reads97.897.89999999999999
Paired Reads137766628138223394
Paired Reads (QC-failed)00
Read16888331469111697
Read1 (QC-failed)00
Read26888331469111697
Read2 (QC-failed)00
Properly Paired Reads131897358132182046
Properly Paired Reads (QC-failed)00
% Properly Paired Reads95.795.6
With itself134451308134992754
With itself (QC-failed)00
Singletons293850284285
Singletons (QC-failed)00
% Singleton0.20.2
Diff. Chroms12320593298
Diff. Chroms (QC-failed)00

Marking duplicates (filtered BAM)

rep1rep2
Unpaired Reads00
Paired Reads5868037758291029
Unmapped Reads00
Unpaired Duplicate Reads00
Paired Duplicate Reads39278593419112
Paired Optical Duplicate Reads00
% Duplicate Reads6.69365.8656

Filtered with samtools flag 1804 (samtools view -F 1804):


Fraction of mitochondrial reads (unfiltered BAM)

rep1rep2
Rn = Number of Non-mitochondrial Reads134092785134669768
Rm = Number of Mitochondrial Reads807975748982
Rm/(Rn+Rm) = Frac. of mitochondrial reads0.00598940287660351240.005530858909862925

SAMstat (filtered/deduped BAM)

rep1rep2
Total Reads109277812109525878
Total Reads (QC-failed)00
Duplicate Reads00
Duplicate Reads (QC-failed)00
Mapped Reads109277812109525878
Mapped Reads (QC-failed)00
% Mapped Reads100.0100.0
Paired Reads109277812109525878
Paired Reads (QC-failed)00
Read15463890654762939
Read1 (QC-failed)00
Read25463890654762939
Read2 (QC-failed)00
Properly Paired Reads109277812109525878
Properly Paired Reads (QC-failed)00
% Properly Paired Reads100.0100.0
With itself109277812109525878
With itself (QC-failed)00
Singletons00
Singletons (QC-failed)00
% Singleton0.00.0
Diff. Chroms00
Diff. Chroms (QC-failed)00

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

rep1rep2
Fraction of reads in NFR0.33908789002965750.41301047624114595
Fraction of reads in NFR (QC pass)FalseTrue
Fraction of reads in NFR (QC reason)out of range [0.4, inf]OK
NFR / mono-nuc reads0.55748784963137850.7639725034037768
NFR / mono-nuc reads (QC pass)FalseFalse
NFR / mono-nuc reads (QC reason)out of range [2.5, inf]out of range [2.5, inf]
Presence of NFR peakTrueTrue
Presence of Mono-Nuc peakTrueTrue
Presence of Di-Nuc peakTrueTrue

rep1
rep1
rep2
rep2

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.



Sequence quality metrics (filtered/deduped BAM)

rep1
rep1
rep2
rep2

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.


Annotated genomic region enrichment

rep1rep2
Fraction of Reads in universal DHS regions0.42955420813147320.4208899562530784
Fraction of Reads in blacklist regions0.0035982235808308460.0034514948147687983
Fraction of Reads in promoter regions0.070734386592586610.06644051737252452
Fraction of Reads in enhancer regions0.355496475350366660.3512251323837824

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.


Library complexity quality metrics


Library complexity (filtered non-mito BAM)

rep1rep2
Total Fragments5844780858074103
Distinct Fragments5470056954824302
Positions with Two Read29023652513223
NRF = Distinct/Total0.9358870.94404
PBC1 = OneRead/Distinct0.9429720.950882
PBC2 = OneRead/TwoRead17.77209120.742868

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.


Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally


Replication quality metrics


IDR (Irreproducible Discovery Rate) plots

rep1_vs_rep2
rep1_vs_rep2
rep1-pr1_vs_rep1-pr2
rep1-pr1_vs_rep1-pr2
rep2-pr1_vs_rep2-pr2
rep2-pr1_vs_rep2-pr2
pooled-pr1_vs_pooled-pr2
pooled-pr1_vs_pooled-pr2

Reproducibility QC and peak detection statistics

overlapidr
Nt193004133410
N1150445104006
N213985894177
Np193846135050
N optimal193846135050
N conservative193004133410
Optimal Setpooled-pr1_vs_pooled-pr2pooled-pr1_vs_pooled-pr2
Conservative Setrep1_vs_rep2rep1_vs_rep2
Rescue Ratio1.00436260388385731.0122929315643505
Self Consistency Ratio1.07569820818258521.1043673083661616
Reproducibility Testpasspass

Reproducibility QC


Number of raw peaks

rep1rep2
Number of peaks264873258482

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics


Peak region size

rep1rep2idr_optoverlap_opt
Min size150.0150.0150.0150.0
25 percentile224.0210.0454.0375.0
50 percentile (median)350.0324.0691.0566.0
75 percentile624.0582.01016.0875.0
Max size3322.02554.03471.03471.0
Mean479.5627678170293450.2582384846914775.3431988152536669.8014403186035

rep1
rep1
rep2
rep2
idr_opt
idr_opt
overlap_opt
overlap_opt

Enrichment / Signal-to-noise ratio


TSS enrichment (filtered/deduped BAM)

rep1rep2
TSS enrichment7.6923821516861177.354598361825624

rep1
rep1
rep2
rep2

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/


Jensen-Shannon distance (filtered/deduped BAM)

rep1rep2
AUC0.264167863773327070.27191882248032284
Synthetic AUC0.495467110274488530.49546942218720447
X-intercept0.098416710324776350.09805478126287248
Synthetic X-intercept0.00.0
Elbow Point0.62910269587160920.6162232202488312
Synthetic Elbow Point0.506230957335280.506253689441241
Synthetic JS Distance0.31841990506590780.3046303531126913

Peak enrichment


Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

rep1rep2rep1-pr1rep2-pr1rep1-pr2rep2-pr2pooledpooled-pr1pooled-pr2
Fraction of Reads in Peaks0.188656714686051720.17340383247144570.171151468515859380.157744416205557990.1710995641091350.157234515065645310.191816106940426830.18159897411603090.1817044418373789

FRiP for overlap peaks

rep1_vs_rep2rep1-pr1_vs_rep1-pr2rep2-pr1_vs_rep2-pr2pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks0.162780120390108580.152350909075668530.136470405651530120.16303710417315173

FRiP for IDR peaks

rep1_vs_rep2rep1-pr1_vs_rep1-pr2rep2-pr1_vs_rep2-pr2pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks0.139610067819240150.127252941338173940.111988885402954720.14051592091522772

For macs2 raw peaks:


For overlap/IDR peaks: